Dual-labeled probes (DLPs) include polynucleotides labeled at one end with fluorescent dye (reporter) and on the opposite end with a quencher molecule. Such probes are often used in various hybridization assays including real-time PCR. In methods that employ DLP(s), when the quencher of the probe is physically close to the reporter, the fluorescence is quenched by physicochemical mechanisms of which FRET (Fluorescence Resonance Energy Transfer) is most common.
The quenching efficiency of a quencher can decrease rapidly with increasing distance between the reporter and the quencher. The distance between the quencher and the reporter molecules in a single free (unbound) intact DLP molecule can, in part, be dependent on the secondary structure of the DLP which is dependent upon the probe sequence. Those DLPs with more secondary structure may exist in a super-coiled conformation where the quencher and reporter are in close proximity and the quenching efficiency is high. Those DLPs with little or no secondary structure are predicted to exist in more relaxed conformation where the quencher and reporter are further apart and the quenching efficiency is lowered. DLPs with lower quenching efficiency can have high fluorescent background because some fluorescence of the reporter ‘escapes’ the effect of the quencher. High fluorescent background is undesirable in real-time PCR assays leading to, for example, low signal to noise ratio resulting in low assay sensitivity or signal drift whereby fluorescent signal from negative samples cross the threshold. When signal from an investigated sample crosses the threshold, a CT value is assigned to it. In automated data analysis where sample discrimination is based on CT values, negative samples displaying signal drift may be classified as positive causing unnecessary re-work.
One way of circumventing DLPs with high fluorescent background is to use attached universal duplex probes (AUDP), as reported in the literature, instead of DLPs. In AUDP, the reporter and quencher are attached respectively to the 5′ and 3′ ends of 2 different molecules of the probe complex which bind to each other. Thus the reporter and quencher are always in close proximity until the probe bearing the quencher is displaced and fluorescence is released. However because of their universal nature, the AUDP do not offer the additional specificity gate for PCR assays that DLPs provide.
There is a need to overcome the limitations, discussed above, of conventional hybridization probes and assays, in particular to provide compositions, methods, and kits that are effective for enhancing the performance of nucleic acid probes, in particular dual-labeled nucleic acid probes.